Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Full citations of these references can be found throughout the specification. Each of these citations is incorporated herein by reference as though set forth in full.
The study of ubiquitin pathway enzymes is hampered by the nature of ubiquitin modification. Ubiquitin (Ub) is transferred from a thioester linkage with the cysteine residue in the active site of a ubiquitin ligase (E2 or E3) to the ε-amine of a lysine residue in the target protein to form an isopeptide bond between the α-carboxyl of the C-terminal glycine of Ub and this amine. The requirement to form an isopeptide linkage places limits on the ability to mimic this process in vitro. Proteases, like most other enzymes, show a high degree of substrate specificity. This can range from relatively low stringency (requiring one of a few specific amino acids at the site of cleavage (the scissile bond)) to medium stringency (requiring a short, specific sequence surrounding the scissile bond) to high stringency (requiring recognition of structurally encoded sites on the target protein away from the scissile bond). This latter class can be exemplified by the ubiquitin and ubiquitin-like protein (UbL) deconjugating enzymes. These DUBs recognize both ubiquitin and the isopeptide bond which they cleave to release free Ub and the protein substrate. All currently available, high throughput compatible assays for DUB activity are based on the cleavage of a linear peptide- or amide-bond, while none measure true isopeptidase activity. In the past, linear peptide libraries have been used extensively to define the substrate specificity resulting from interactions between the side chains of residues surrounding the scissile bond and sub-sites within the active site of a protease for a wide variety of proteases including caspases, cathepsin S, and γ-secretase. The nature of the side chains and their interactions with the active site have been essential for guiding structure based drug design of small molecule inhibitors. These kinds of studies cannot be carried out with DUBs because linear peptides do not reflect the geometry of an isopeptide bond.
There are currently three commercially available assays for measuring the activity of Ub/Ub1 isopeptidases. The first assay consists of the Ub/Ub1 fused to a fluorophore, typically amino-methyl coumarin (AMC; FIG. 1A). Upon incubation with an isopeptidase, AMC is released and is associated with a detectable increase in fluorescence (Dang et al. (1998) Biochem., 37:4868-4879). The second assay is the Ubiquitin LanthaScreen™ reagent available from Invitrogen (Carlsbad, Calif.; U.S. Patent Application Publication No. 2007/0264678). This assay measures fluorescence resonance energy transfer between a fluorophore at the N-terminus of ubiquitin and a second fluorophore at the C-terminus. Incubation with an isopeptidase causes release of the fluorophore at the C-terminus of ubiquitin and a loss of fluorescence resonance energy transfer (FRET) signal. In addition to being a “loss of signal” readout, a major disadvantage of this assay format is the use of non-physiological substrates, given the observation that perturbations at the N-terminus of ubiquitin impact ubiquitin structure/function. The third assay is the CHOP reporter system (LifeSensors, Inc. (Malvern, Pa.); www.lifesensors.com; U.S. Patent Application Publication No. 2006/0040335). The substrate in this assay is an Ub-PLA2 fusion from which an active PLA2 enzyme is released upon incubation with an isopeptidase. Active PLA2 cleaves a quenched fluorescent substrate causing an increase in fluorescence. This assay is extremely sensitive and works well with many members of the USP family, yet this assay still relies on cleavage of an amide bond within the substrate and is therefore not as physiologically relevant as diubiquitin.
While researchers wishing to measure Ub/Ub1 isopeptidase activity with a physiological substrate can obtain ubiquitin chains and detect the cleavage of these chains by SDS-PAGE followed by Western blotting with an antibody recognizing Ub/Ub1, this method is low throughput and time intensive. Moreover, kinetic parameters cannot be determined in such experiments.
Gururaja et al. have reported a fluorescence based homogeneous assay for monitoring multi-Ub chain assembly and disassembly (Gururaja et al. (2005) Methods Enzymol., 399:663-682). The assay is based on the use of internally quenched fluorescent pairs. To add the quencher and fluorophore, the N-terminal methionine was changed to a cysteine. Additionally, a FLAG tag was added. Polymerization was carried out by using all the three enzymes (E1, E2 and E3) involved in conjugation and the quenching of fluorescence was monitored. Once the polyubiquitin was synthesized, the reaction was stopped and a DUB was added to monitor deubiquitylase activity. The assay had multiple components that required considerable standardization. Moreover, based on the crystal structure of ubiquitin, Vijay-Kumar et al. have proposed that the N-terminus of ubiquitin is virtually inaccessible (Vijay-Kumar et al. (1987) J. Mol. Biol., 194:531-544). Therefore, modification of the N-terminus interferes with the formation and recognition of diubiquitin. In fact, attaching a tag at the N-terminus of Ub has been known to interfere with the formation of K63-linked diubiquitins (Pickart et al. (2005) Methods Enzymol., 399:21-36). Development of this substrate requires multiple enzymes. Additionally, modifying the N-terminus of ubiquitin alters the structure of the protein and, thus, affect potential interactions with a DUB.
U.S. Patent Application Publication No. 2004/0053324 also describes the use of a FRET-based system to assay deubiquitinating activity. In a specific example of deubiquitinating activity, the FRET pair used is fluorescein and tetramethylaminorhodamine (TAMRA). As with Gururaja et al., the ubiquitin moiety is modified to have an N-terminal FLAG tag and an additional cysteine residue for linkage of the fluorescein. No other specific locations in the ubiquitin molecule for the placement of a fluorophore for the optimal assay of isopeptidase activity are described.
The mechanism by which certain Ub/Ub1-specific proteases recognize and cleave their cognate Ub/Ub1 substrate (“specificity”) is not uniform. At the gene level, ubiquitin is encoded as a head-to-tail linked poly-ubiquitin (6-15 units of monomer Ub arranged in a head-to-tail fashion). Ubiquitin is also encoded as monomers linked to a C-terminal extension, such as a Ub-ribosomal fusion protein. In order for ubiquitin to enter the ubiquitinylation pathway and for conjugation of the C-terminus of ubiquitin to target proteins, linear poly-ubiquitin or ubiquitin carboxyl extension proteins must be cleaved by DUBs to form mature ubiquitins. Among the ˜100 DUBs encoded by the human genome, selected DUBs are responsible for the generation of free ubiquitin to enter the ubiquitin pathway. Ub1s, such as SUMO, are also encoded at the gene level in precursor form. Thus, nature has designed certain DUBs that recognize Ub and Ub1 C-terminal peptide extensions as substrates. In these cases, specificity is thought to be determined by discrete interactions between the protease and the amino acid residues in and around the active site. This assumption of specificity has led to the wide spread use of Ub/Ub1 conjugates that have a small adjunct (typically fluorescent in nature) linked to a C-terminus peptide. While these conjugates are cleaved to a measurable degree by some Ub/Ub1 specific proteases (such as UCHL3 (ubiquitin carboxyl-terminal hydrolase isozyme L3), SENP2 (sentrin-specific protease 2), PLpro (papain-like protease)), other Ub/Ub1-specific proteases exhibit no detectable activity towards these reporter molecules (such as Otubain2 (Otub2), AMSH (associated molecule with the SH3 domain of STAM), JosD1 (Josephin-1)). The activity of enzymes such as Otub2, AMSH, and JosD1 can be detected by the cumbersome and time consuming monitoring of polyubiquitin degradation by immunoblotting. Moreover, kinetic parameters cannot be determined from such assay formats. Presumably, the poor reactivity of Ub/Ub1 C-terminal adducts with certain proteases is related to specificity requirements beyond discrete interactions with amino acids surrounding the bond to be cleaved. Considering that the majority of known Ub/Ub1-specific proteases (100+) have not been adequately characterized with respect to relative activity or specificity, there exists a true need for novel reagents for the characterization of these isopeptidases. It is also desirable to provide an assay for isopeptidases that uses a physiologically relevant substrate and that generates a powerful signal of protease activity.
In view of the foregoing, there is a clear need for a better fluorescent method for detecting Ub isopeptidase activity using a physiologically relevant substrate which will also be useful in high-throughput screening (e.g., for drug discovery).